Isoelectric focusing is a useful technique for separating proteins and polypeptides in solution. This technique utilizes the fact that such compounds are zwitterions and thus have a definite pH at which they carry no net charge (isoelectric point). At their isoelectric point proteins and polypeptides will not migrate in an electric field. It has thus been possible to separate proteins and polypeptides by establishing a pH gradient with small molecular weight zwitterions (carrier ampholytes) in an isoelectric focusing device since the compunds will migrate to their characteristic isoelectric point and stop in such a gradient.
However, there have been restrictions on the size of samples which can be separated by previously available devices. The smallest devices required at least about 20 ml. of sample volume. Such volumes are too large when only small amounts of the protein or polypeptide are available since dilution would result in unacceptably low concentrations for analysis.
Smaller volume devices were particularly prone to problems arising from the phenomenon of electro-osmotic flow. This relatively large volume flow of water from one electrode to the other will cause the compounds being separated to migrate from their true isoelectric points. When the compound has a relatively weak charge density such migration can be quite extensive until the charge on the compound provides an electrostatic force sufficient to offset the force of the osmotic flow. In addition, the osmotic flow will occur mainly in the center of the channel and will fall off rapidly towards the sides. This will cause the concentration gradient to be bullet shaped and thus will not be normal to the long axis as desired for maximum resolution. Such distortion is proportionately greater in the smaller volume devices and thus prevents such small volume devices to function effectively as an analytical or preparative instrument.
One method which has been employed in the art to separate small amounts of proteins or polypeptides is to utilize a stationary support phase for the compound solution. Thus only a thin liquid film is present and the effects of electro-osmotic flow are not critical. However, these support materials such as acrylamide gel, sephadex and the like are quite expensive. Furthermore, separations on such devices are comparatively slow and recovery of the separated compounds is either laborious or not possible at all.